A cell digestion solution, a method for isolating gamma delta T cells and applications

Abstract

This invention relates to the medical field, specifically to a cell digestion solution, a method for isolating γδT cells, and its application. The cell digestion solution comprises: collagenase, mammalian serum, antibiotics, a buffer, and a cell culture medium; wherein the collagenase content is 1-4 mg / ml, the mammalian serum content is 5 vol%-15 vol%, the antibiotic content is 0.5 vol%-2 vol%, and the buffer content is 5-15 mM / L. Through the synergistic effect and optimized content of components such as collagenase, mammalian serum, and buffer, the cell digestion solution effectively protects cell viability and surface antigen integrity while efficiently dissociating skin tissue, providing a high-quality cell source for subsequent isolation of rare immune cells.

Description

Technical Field

[0001] This invention relates to the medical field, and more specifically, to a cell digestion solution, a method for separating γδT cells, and its application. Background Technology

[0002] The skin, the largest organ in the human body, possesses a variety of physiological functions, including protection, sensation, temperature regulation, excretion, and immunity. The immune microenvironment plays a crucial role in many physiological and pathological processes. Diseases such as eczema, acne, psoriasis, contact dermatitis, and skin-related tumors (e.g., melanoma) are all closely related to the functional regulation of local immune cells in the skin. Mouse models are important tools for studying the occurrence, development, and intervention strategies of diseases (especially skin diseases). In this research system, the efficient and highly active isolation of specific immune cell subsets in skin tissue is a prerequisite for in-depth analysis of their functions and mechanisms.

[0003] γδ T cells are a subset of T cells that lie between innate and adaptive immunity, primarily distributed in the mucous membranes and subcutaneous tissues of the skin. This cell subset participates in immune regulation through functions such as secreting cytokines (e.g., IFN-γ and IL-17) and exerting cytotoxic effects, playing a crucial role in skin infection defense, tumor immune surveillance, and inflammation control. Recent studies have shown that γδ T cells exhibit active immunomodulatory properties in diseases such as psoriasis, atopic dermatitis, cutaneous lymphoma, and melanoma, making them a potential target for skin immunotherapy research. Unlike keratinocytes and fibroblasts, γδ T cells are present in extremely low proportions and are mostly distributed around hair follicles, in the basal layer of the epidermis, and at the dermal-epidermal junction, tightly encapsulated by keratinocytes, collagen fibers, and the extracellular matrix. Traditional enzymatic digestion methods struggle to achieve efficient release while preserving cell viability.

[0004] Currently, the methods commonly used for preparing single-cell suspensions of mouse skin often employ a single collagenase or a combination of collagenase and deoxyribonuclease digestion. However, these methods often suffer from problems such as low efficiency or complex operation, especially for rare cell subpopulations such as γδ T cells, where efficiency is even lower.

[0005] In conclusion, developing a digestive system that can efficiently and effectively release γδ T cells from skin tissue has significant technical value and application prospects for advancing skin immunology research and elucidating the mechanisms of related disease models.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] To address the problems of low efficiency, cumbersome procedures, and unsatisfactory cell viability in existing skin γδT cell isolation technologies, this invention provides a cell digestion solution, a method for separating γδT cells, and its application. The digestion system and separation process provided by this invention significantly improve the yield and viability of γδT cells. The operation is simple and time-saving, greatly improving the separation efficiency. It can be widely used in basic research on skin immunity, pathogenesis of skin diseases, anti-infection and anti-tumor immunity, and immune regulation research and development. It also provides a reliable reference for the separation of other trace cells in the skin, helping to conduct subsequent research more efficiently.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: A cell digestion solution includes: collagenase, mammalian serum, antibiotics, buffers, and cell culture medium; wherein the collagenase content in the cell digestion solution is 1-4 mg / ml, the mammalian serum content is 5 vol%-15 vol%, the antibiotic content is 0.5 vol%-2 vol%, and the buffer content is 5-15 mM / L.

[0009] The cell digestion solution, through the synergistic effect and optimized content of components such as collagenase, mammalian serum, and buffer, can effectively protect cell viability and surface antigen integrity while efficiently dissociating skin tissue, providing a high-quality cell source for the subsequent isolation of rare immune cells.

[0010] A method for isolating γδT cells includes the following steps: (a) incubating skin tissue in an isotonic equilibrium solution, performing a first vortex and a first filtration to collect skin tissue fragments; (b) washing the skin tissue fragments, performing a second filtration and pulverizing; mixing the pulverized skin tissue fragments with the cell digestion solution and performing digestion treatment, performing a second vortex and a third filtration, centrifuging the filtrate, discarding the supernatant, and obtaining a cell precipitate; (c) resuspending the cell precipitate using a first density gradient centrifugation medium; placing the resuspended cell resuspended solution on a second density gradient centrifugation medium, performing density gradient centrifugation, and aspirating the cell layer at the interface between the first and second density gradient centrifugation media.

[0011] The described method for isolating γδT cells significantly improves the yield and viability of γδT cells through synergistic optimization of steps such as pretreatment of loose tissue, gentle shaking digestion, and discontinuous density gradient centrifugation. This method is simple to operate, time-efficient, and the resulting cells can be widely used in basic research on skin immunity, exploration of skin disease mechanisms, and screening of immunomodulatory drugs.

[0012] The use of the cell digestion solution described above in the preparation of a kit for isolating skin γδT cells.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The cell digestion solution provided by this invention achieves both efficient digestion of skin tissue and protection of cell viability through the synergistic effect of specific components and their concentration ranges. Specifically, collagenase specifically degrades collagen fibers in the extracellular matrix of skin cells; albumin and antiprotease components in mammalian serum effectively neutralize protease toxicity during digestion, protecting cell membrane integrity; antibiotics prevent microbial contamination; buffers stably maintain the physiological pH range in the air environment; and cell culture medium provides basic nutritional support. Through the optimized ratio of its components, this digestion solution ensures efficient tissue dissociation while maximizing the preservation of target cell viability, making it particularly suitable for the subsequent isolation of rare immune cells (such as γδT cells), providing a high-quality cell source for skin immunology research.

[0014] (2) The γδT cell isolation method provided by this invention achieves a technological breakthrough in the efficient and highly active isolation of rare γδT cells from skin tissue through multi-step synergistic optimization. The method first pre-treats the cells with an isotonic equilibrium solution containing chelating agents and buffers to remove calcium and magnesium ions and loosen the tissue; then, it uses an optimized cell digestion solution under gentle shaking conditions for digestion, precisely controlling the temperature, time, and shaking frequency to release cells while protecting surface antigens; finally, it uses discontinuous density gradient centrifugation, utilizing first and second density gradient centrifugation media of specific concentrations and volume ratios, to precisely enrich γδT cells at the medium interface under optimized centrifugation conditions. This method is simple to operate, time-saving, and yields high-yield and highly active γδT cells. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 This is a graph showing the flow cytometry results from Example 5. Figure 2 This is a flow cytometry cell population ratio statistical analysis graph from Example 5. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0018] One aspect of the present invention relates to a cell digestion solution comprising: collagenase, mammalian serum, antibiotics, buffers, and cell culture medium; In the cell digestion solution, the collagenase content is 1~4 mg / ml (e.g., it can be any one of 1 mg / ml, 2 mg / ml, 3 mg / ml or 4 mg / ml or a range between any two), the mammalian serum content is 5 vol%~15 vol% (e.g., it can be any one of 5 vol%, 7 vol%, 9 vol%, 11 vol%, 13 vol% or 15 vol% or a range between any two), the antibiotic content is 0.5 vol%~2 vol% (e.g., it can be any one of 0.5 vol%, 1 vol%, 1.5 vol% or 2 vol% or a range between any two), and the buffer content is 5~15 mM / L (e.g., it can be any one of 5 mM / L, 7 mM / L, 9 mM / L, 11 mM / L, 13 mM / L or 15 mM / L or a range between any two).

[0019] The cell digestion solution described herein utilizes collagenase to specifically degrade collagen fibers in skin tissue, while simultaneously protecting cell membrane integrity with albumin and antiprotease components from mammalian serum, preventing microbial contamination, maintaining pH stability of the digestion solution with a buffer, and providing basic nutrition with cell culture medium. The synergistic effect of these components ensures efficient release of skin cells while maintaining high cell activity, laying the foundation for subsequent isolation of rare γδT cells.

[0020] Furthermore, in the cell digestion solution, the content of collagenase is 1.5~3.5 mg / ml, the content of mammalian serum is 8 vol%~13 vol%, the content of antibiotic is 1 vol%~1.8 vol%, and the content of buffer is 8~13 mM / L.

[0021] Furthermore, the collagenase includes at least one of type I collagenase, type II collagenase, type IV collagenase, or type XI collagenase, preferably type IV collagenase. The collagenase can be selected from any one of the above collagenases, or a combination of any two, or a combination of any three; multiple collagenases can be combined in any ratio.

[0022] Type IV collagenase primarily degrades type IV collagen with less damage to cells. It is usually extracted from Clostridium histolytica and is suitable for epithelial tissues, fibrous connective tissues, etc.

[0023] Furthermore, the mammalian serum includes at least one of fetal bovine serum, newborn calf serum, or horse serum. The albumin, anti-protease components, and growth factors in the serum can neutralize the toxicity of proteases released during digestion, stabilize cell membrane structure, and provide basic nutrition, thereby protecting the integrity of γδT cell surface antigens (such as γδTCR) and maintaining cell viability.

[0024] Furthermore, the antibiotics include penicillin and / or streptomycin, added in solution form at concentrations conventional in the art. Penicillin inhibits Gram-positive bacteria, and streptomycin inhibits Gram-negative bacteria; the combined use of the two can effectively prevent bacterial contamination, ensure a sterile environment during the isolation process, and avoid interference from microorganisms with cell viability and subsequent experiments.

[0025] Furthermore, the buffer comprises any one of HEPES, TES, or MOPS, preferably HEPES. These zwitterionic buffers can stably maintain the pH within the physiological range in an air environment, preventing pH fluctuations caused by acid production from cell metabolism or CO2 escape, thereby protecting cell activity and ensuring the stability of the digestion and separation process.

[0026] HEPES stands for 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid. It is a buffer solution based on the principle of organic sulfonic acid buffer pairs. It is designed to provide a stable and non-toxic buffer environment within the physiological pH range (pH 6.8 - 8.2) and can maintain a stable pH in systems lacking a stable CO2 environment.

[0027] Furthermore, the cell culture medium includes any one of RPMI-1640 medium, DMEM medium, or IMDM medium, preferably RPMI-1640 medium. These classic culture media can provide the amino acids, vitamins, inorganic salts, and energy substrates required for short-term cell survival, maintain osmotic pressure balance, provide basic nutritional support for cells during digestion, prevent cell death due to nutrient deficiency, and improve the final cell yield.

[0028] One aspect of the present invention also relates to a method for isolating γδ T cells, comprising the following steps: (a) Skin tissue is incubated in an isotonic equilibrium solution, subjected to a first vortex and a first filtration, and skin tissue fragments are collected; (b) The skin tissue fragments are washed, filtered a second time, and pulverized; the pulverized skin tissue fragments are mixed with the cell digestion solution and then digested, vortexed a second time, and filtered a third time. The filtrate is centrifuged, the supernatant is discarded, and cell precipitate is obtained. (c) The cell pellet is resuspended using a first density gradient centrifugation medium; the resuspended cell suspension is placed on a second density gradient centrifugation medium and centrifuged to collect the cell layer at the junction of the first density gradient centrifugation medium and the second density gradient centrifugation medium.

[0029] The described γδT cell isolation method, through multi-step synergistic optimization, achieves a technological breakthrough in the efficient and highly active isolation of rare γδT cells from skin tissue. The method first pre-treats the tissue with an isotonic balanced solution containing chelating agents and buffers to remove calcium and magnesium ions and loosen the tissue. Then, an optimized cell digestion solution is used for digestion under gentle shaking conditions, with precise control of temperature, time, and shaking frequency to release cells while protecting surface antigens. Finally, discontinuous density gradient centrifugation is performed using first and second density gradient centrifugation media of specific concentrations and volume ratios under optimized centrifugation conditions to precisely enrich γδT cells at the medium interface. This method is simple to operate, time-efficient, and yields high-yield and highly active γδT cells.

[0030] Furthermore, the skin tissue is pretreated skin tissue, the pretreatment including: scraping off scales from the surface of the skin tissue, removing fat and connective tissue from the inner surface of the skin tissue, and cutting the skin tissue into pieces of 0.3~0.7 cm. Skin fragments of 0.3~0.7 cm.

[0031] Further, the isotonic equilibrium solution comprises: a basal balanced salt solution, a chelating agent at a concentration of 2–8 mM / L (e.g., a point value or a range between any two of 2 mM / L, 4 mM / L, 6 mM / L, or 8 mM / L), and the buffer at a concentration of 8–12 mM / L (e.g., a point value or a range between any two of 8 mM / L, 9 mM / L, 10 mM / L, or 12 mM / L). The chelating agent, by binding calcium and magnesium ions, disrupts calcium-dependent adhesion between cells, loosening the tissue; the buffer maintains pH stability; and the basal balanced salt solution provides an isotonic environment. These three components work synergistically to fully loosen the skin tissue during the pretreatment stage, creating favorable conditions for subsequent enzymatic digestion.

[0032] Furthermore, the basic balanced salt solution includes HBSS, in which calcium and magnesium ions have been removed. HBSS itself provides an isotonic environment, while the removal of calcium and magnesium ions completely breaks down intercellular connections, preventing divalent cations from interfering with subsequent collagenase activity, while also reducing cell aggregation and improving digestion efficiency. HBSS acts as a buffer and contains glucose to nourish cells.

[0033] Furthermore, the chelating agent includes EDTA.

[0034] Furthermore, the incubation temperature is 35-39°C (e.g., any value or range between any two of 35°C, 36°C, 37°C, 38°C, or 39°C), and the time is 25-35 min (e.g., any value or range between any two of 25 min, 27 min, 29 min, 31 min, 33 min, or 35 min), and the incubation is carried out under shaking conditions at a frequency of 190-210 r / min (e.g., any value or range between any two of 190 r / min, 195 r / min, 200 r / min, 205 r / min, or 210 r / min). Suitable temperature promotes the action of chelating agents and buffers, while gentle shaking accelerates liquid circulation, ensuring sufficient contact between the tissue and the pretreatment solution, effectively loosening the tissue, and avoiding cell damage due to excessive shaking.

[0035] Furthermore, the duration of the first vortex oscillation is 12 to 18 seconds, including but not limited to a point value of any one of 12 seconds, 14 seconds, 16 seconds, or 18 seconds, or a range between any two.

[0036] Furthermore, the pore size of the filter medium in the first filter is 70~100μm, including but not limited to a point value of any one of 70μm, 80μm, 90μm or 100μm or a range between any two. This pore size can effectively trap large, unloosened tissue fragments while allowing loosened cells and small tissue fragments to pass through, ensuring that the tissue fragments in subsequent processing are of uniform size and facilitate further digestion.

[0037] Furthermore, the first filtering is repeated 2 to 3 times.

[0038] Furthermore, the washing solution includes PBS.

[0039] Furthermore, the pore size of the filter medium in the second filtration is 90~110μm, including but not limited to a point value of any one of 90μm, 95μm, 100μm, 105μm or 110μm or a range between any two. This pore size is used to remove incompletely digested tissue fragments remaining after digestion, ensuring the purity of the cell suspension in the filtrate and reducing interference from impurities in subsequent centrifugation steps.

[0040] Furthermore, the size of the pulverized skin tissue fragments is 1-2 mm. 1~2 mm.

[0041] Furthermore, the mixing ratio of the pulverized skin tissue fragments and the cell digestion fluid is 1~4 mm. 2 10mL, including but not limited to 1mm 2 10mL, 2mm 2 10mL, 3mm 2 10mL or 4mm 2 10 mL. This ratio ensures sufficient digestive fluid to cover tissue fragments, preventing tissue overload and incomplete digestion, while also preventing waste of digestive fluid, ensuring optimal contact between enzymes and substrates, and improving cell release efficiency.

[0042] Furthermore, the digestion process is carried out at a temperature of 35–39°C (e.g., any value or range between any two of 35°C, 36°C, 37°C, 38°C, or 39°C), for a time of 28–32 min (e.g., any value or range between any two of 28 min, 29 min, 30 min, 31 min, or 32 min), and under shaking conditions at a frequency of 190–210 r / min (e.g., any value or range between any two of 190 r / min, 195 r / min, 200 r / min, 205 r / min, or 210 r / min). This temperature range is optimal for collagenase activity. Gentle shaking promotes enzyme-tissue contact, promptly disperses the released cells into the solution, and avoids over-digestion that could damage cell surface antigens, thereby maintaining high activity while efficiently releasing cells.

[0043] Furthermore, the duration of the second vortex oscillation is 12 to 18 seconds, including but not limited to a point value of any one of 12 seconds, 14 seconds, 16 seconds, or 18 seconds, or a range between any two.

[0044] Furthermore, the pore size of the filter medium in the third filter is 70~100μm, including but not limited to a point value of any one of 70μm, 80μm, 90μm or 100μm or a range between any two. This further fine filtration removes minute impurities and cell clumps, obtaining a single-cell suspension, providing a high-quality cell sample for subsequent density gradient centrifugation.

[0045] Furthermore, the centrifugal force is 345-355g (e.g., any value or range between any two of 345g, 347g, 349g, 351g, 353g, or 355g), and the time is 4-6min (e.g., any value or range between any two of 4min, 5min, or 6min). This centrifugal force effectively precipitates cells while avoiding excessive centrifugal force that could cause cell damage or activation. The appropriate time ensures a high cell recovery rate, and the supernatant is discarded to obtain a precipitate rich in live cells.

[0046] Furthermore, the concentration of the first density gradient centrifugation medium is 35 vol% to 45 vol%, including but not limited to point values ​​of any one of 35 vol%, 37 vol%, 39 vol%, 41 vol%, 43 vol%, or 45 vol%, or a range between any two. This concentration corresponds to a density lower than that of lymphocytes. After centrifugation, dead cells and debris remain in this layer, while lymphocytes settle to the lower interface, thus achieving preliminary purification.

[0047] Furthermore, the concentration of the second density gradient centrifugation medium is 70 vol% to 75 vol%, including but not limited to point values ​​of any one of 70 vol%, 71 vol%, 72 vol%, 73 vol%, 74 vol%, or 75 vol%, or a range between any two. This concentration corresponds to a density higher than that of lymphocytes but lower than that of keratinocytes, fibroblasts, and erythrocytes. Therefore, lymphocytes are blocked at the interface between the two media layers, while high-density cells sink to the bottom of the tube, effectively enriching lymphocytes.

[0048] A density gradient centrifugation medium conventional in this field can be used, such as, but not limited to, percoll.

[0049] In some specific implementations, the preparation methods for 72% percoll and 40% percoll are as follows: Take 18ml of 100% percoll and add 2ml of 10X PBS to obtain 20ml of 90% percoll-1. Take 8.9ml of percoll-1 and add 11.1ml of RPMI-1640 medium to obtain 20ml of 40% percoll-2. Similarly, take 8.6ml of percoll-1 and add 2.4ml of RPMI-1640 medium to obtain 12ml of 72% percoll-3.

[0050] Furthermore, the volume ratio of the first density gradient centrifugation medium to the second density gradient centrifugation medium is 3 to 5:2, including but not limited to 3:2, 4:2, or 5:2. This volume ratio forms a stable discontinuous density gradient, ensuring a clear interface between the two media layers during centrifugation, allowing for thorough cell separation, avoiding interface blurring or cell mixing due to improper volume ratios, and improving the purity of lymphocyte recovery.

[0051] Furthermore, the density gradient centrifugation speed is 2150~2250 r / min (e.g., any value or range between any two of 2150 r / min, 2170 r / min, 2190 r / min, 2210 r / min, 2230 r / min, or 2250 r / min), and the time is 18~22 min (e.g., any value or range between any two of 18 min, 19 min, 20 min, 21 min, or 22 min). This centrifugation speed generates sufficient centrifugal force to allow cells to migrate to their corresponding locations according to density, while the time is moderate, ensuring sufficient cell separation without causing cell compression damage due to excessive centrifugation. Combined with minimum acceleration and deceleration settings, interface stability can be effectively maintained, improving the yield and purity of γδT cells.

[0052] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0053] Example 1 The cell digestion solution provided in this embodiment includes: collagenase, mammalian serum, antibiotics, buffer, and cell culture medium; The cell digestion solution contained 1.5 mg / ml collagenase, 10 vol% mammalian serum, 1 vol% antibiotics, and 10 mM / L buffers. The collagenase used was type IV collagenase, the mammalian serum was fetal bovine serum, the antibiotics were penicillin and streptomycin, the buffer was HEPES, and the cell culture medium was RPMI-1640 medium.

[0054] Example 2 The only difference between this embodiment and Example 1 is the type of collagenase used; type II collagenase is employed.

[0055] Example 3 The only difference between this embodiment and Embodiment 1 is the type of buffer used; MOPS is used instead.

[0056] Example 4 The only difference between this embodiment and Example 1 is the type of cell culture medium used; DMEM medium is used.

[0057] Example 5 The only difference between this embodiment and Example 1 is the type of collagenase used; a mass ratio of type I collagenase and type XI collagenase is used.

[0058] Example 6 The only difference between this embodiment and Embodiment 1 is the type of mammalian serum used; horse serum is employed.

[0059] Example 7 This embodiment describes a method for isolating a small number of γδT cells from skin tissue of an IMQ-induced psoriasis model and analyzing the proportion of its γδT17 cell subset, including the following steps: 1. Disease model induction: A mouse psoriasis model was induced using IMQ. Day 0: Hair (approximately 2cm) was removed from the backs of mice using animal hair clippers. (3cm); Day 1-Day 6: Apply 62.5mg imiquimod evenly to the back skin of each mouse once daily; observe and record changes in mouse skin phenotype and symptoms, and obtain skin samples on Day 7; 2. Euthanize the mice and use ophthalmic scissors to harvest the required skin tissue (approximately 1 cm). After cleaning, use a scalpel to scrape away surface scales and excess fat and connective tissue from the inner skin; cut the cleaned mouse skin into pieces (approximately 0.5cm). 0.5cm), transfer the shredded skin to a 50ml centrifuge tube, add 10ml of isotonic equilibrium solution preheated to 37℃, the isotonic equilibrium solution includes: HBSS, 5mM EDTA and 10mM HEPES, HBSS contains phenol red and does not contain calcium or magnesium; 3. Place the centrifuge tube containing skin fragments on a shaker and incubate at 37°C and 200 rpm for 30 minutes. The shaker should be a constant temperature shaker with a maximum speed of 350 rpm. 4. At the end of the incubation, vortex the centrifuge tube vigorously for 15 seconds until the supernatant turns milky; filter the contents of the test tube through a 100-micron filter, collect the skin fragments and transfer them to a new 50 ml centrifuge tube, and repeat the above steps; 5. Thoroughly wash the incubated skin tissue with PBS to remove any remaining EDTA, in order to prevent EDTA from chelating zinc ions and causing type IV collagenase inactivation during subsequent digestion steps. 6. Filter the test tube contents again through a 100-micron filter, collect the skin fragments, and mechanically pulverize or cut the skin fragments into 2mm pieces. 2 The cells were sized and transferred to the bottom of a 50 ml centrifuge tube; 10 ml of the cell digestion solution from Example 1, preheated to 37°C, was added; the centrifuge tube containing the cell digestion solution and skin debris was placed on a constant temperature shaker and digested at 37°C and 200 rpm for 30 minutes. After digestion, the skin debris was noticeably smaller and less numerous; at the end of digestion, the centrifuge tube was shaken for 15 seconds using a vortex mixer. 7. Filter the contents of the centrifuge tube through a 70-micron filter and transfer the filtrate to a new 50-milliliter centrifuge tube. Collect the skin fragments and temporarily store the resulting filtrate on ice. 8. Centrifuge the filtrate at 350g for 5 minutes, discard the supernatant, and obtain the cell pellet; 9. Use density gradient centrifugation to separate lymphocytes from a single-cell suspension. Resuspend the cell pellet in 4 ml of 40 vol% percoll. Gently add the 4 ml of 40 vol% percoll containing cells to 2 ml of 72 vol% percoll in a 15 ml centrifuge tube using a pipette. Ensure the pipette is set to the lowest speed and place it along the wall of the centrifuge tube, being careful not to shake, disturb, or invert it. Set the centrifuge acceleration to the lowest setting (0 for initial acceleration, 0 for final acceleration, or maintain the lowest centrifuge acceleration). Centrifuge at 2200 r / min for 20 minutes at room temperature (23℃), ensuring the centrifuge is in a balanced state. 10. After centrifugation, gently transport the 15ml centrifuge tube to the lab bench and place it upright. Using a 5ml plastic transfer pipette, aspirate the cell layer at the boundary between the 40vol% and 72vol% percoll and transfer it to a new 15ml centrifuge tube. If the interface is not visible, simply remove about 2ml of liquid from the boundary between the 40vol% and 72vol% percoll. 11. Add 1x ice-cold PBS containing 2 vol% FBS to a centrifuge tube containing interface cells, centrifuge the cells at 1600 r / min for 6 min, pour off the supernatant, and the obtained cells are the target cells. Perform routine flow cytometry antibody staining on the obtained cells and analyze them.

[0060] Example 8 The only difference between this embodiment and Embodiment 7 is that the cell digestion solution used in Embodiment 2 is employed.

[0061] Example 9 The only difference between this embodiment and embodiment 7 is that the cell digestion solution used in embodiment 3 is employed.

[0062] Example 10 The only difference between this embodiment and embodiment 7 is that the cell digestion solution used in embodiment 4 is employed.

[0063] Example 11 The only difference between this embodiment and Embodiment 7 is that the cell digestion solution used in Embodiment 5 is employed.

[0064] Example 12 The only difference between this embodiment and embodiment 7 is that the cell digestion solution used in embodiment 6 is employed.

[0065] Comparative Example 1 The only difference between this embodiment and Embodiment 1 is the content of the components, as detailed below: The collagenase concentration was 8 mg / ml, the mammalian serum concentration was 2 vol%, and the buffer concentration was 20 mM / L. The separation method described in Example 7 was used.

[0066] Experimental Example Figure 1 The image shown is a flow cytometry result from Example 7, illustrating the flow cytometry gating strategy for mouse skin single-cell suspensions and the proportion of each cell population. The results indicate that the mouse skin single-cell suspensions prepared using this method exhibit good cell viability and a high yield of γδT cells.

[0067] Figure 2 This is a flow cytometry cell population ratio statistical analysis diagram from Example 7, showing live cells in a mouse skin single-cell suspension, CD45 +The proportions of cells, δγT cells, and γδT17 cell populations (five biological replicates achieved). The assay method was flow cytometry.

[0068] Table 1

[0069] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell digest, characterized in that, include: Collagenase, mammalian serum, antibiotics, buffers, and cell culture media; In the cell digestion solution, the collagenase content is 1~4 mg / ml, the mammalian serum content is 5 vol%~15 vol%, the antibiotic content is 0.5 vol%~2 vol%, and the buffer content is 5~15 mM / L.

2. The cell digest of claim 1, wherein, In the cell digestion solution, the collagenase content is 1.5~3.5 mg / ml, the mammalian serum content is 8 vol%~13 vol%, the antibiotic content is 1 vol%~1.8 vol%, and the buffer content is 8~13 mM / L.

3. The cell digest of claim 1 or 2, wherein, The collagenase includes at least one of type I collagenase, type II collagenase, type IV collagenase, or type XI collagenase. And / or, the mammalian serum includes at least one of fetal bovine serum, newborn calf serum, or horse serum.

4. The cell digest of claim 1 or 2, wherein, Includes at least one of the following technical features: (1) The antibiotics include: penicillin and / or streptomycin; (2) The buffer includes any one of HEPES, TES or MOPS; (3) The cell culture medium includes any one of RPMI-1640 medium, DMEM medium or IMDM medium.

5. A method of isolating γδ T cells, characterized in that, Includes the following steps: (a) Skin tissue is incubated in an isotonic equilibrium solution, subjected to a first vortex and a first filtration, and skin tissue fragments are collected; (b) The skin tissue fragments are washed, filtered a second time, and pulverized; the pulverized skin tissue fragments are mixed with the cell digestion solution according to any one of claims 1 to 4 and then digested, vortexed a second time, and filtered a third time; the filtrate is centrifuged, the supernatant is discarded, and cell precipitate is obtained. (c) The cell pellet is resuspended using a first density gradient centrifugation medium; the resuspended cell suspension is placed on a second density gradient centrifugation medium and centrifuged to collect the cell layer at the junction of the first density gradient centrifugation medium and the second density gradient centrifugation medium.

6. The method of isolating γδ T cells according to claim 5, wherein, Includes at least one of the following technical features: (1) The isotonic equilibrium solution comprises: a basic equilibrium salt solution, a chelating agent of 2-8 mM / L and the buffer of 8-12 mM / L; (2) The basic equilibrium salt solution includes: HBSS, wherein calcium and magnesium ions in the HBSS are removed; (3) The incubation temperature is 35~39℃, the incubation time is 25~35min, and the incubation is carried out under shaking conditions with a shaking frequency of 190~210r / min; (4) The pore size of the filter medium of the first filter is 70~100μm.

7. The method of isolating γδ T cells according to claim 5, wherein, Includes at least one of the following technical features: (1) the mixed ratio of the pulverized skin tissue fragments and the cell digestion solution is 1 ~ 4mm 2 : 10 mL; (2) The digestion process is carried out at a temperature of 35~39℃ for 28~32 min, and the digestion process is carried out under shaking conditions with a shaking frequency of 190~210 r / min; (3) The pore size of the filter medium in the second filter is 90~110μm; (4) The pore size of the filter medium of the third filter is 70~100μm.

8. The method of isolating γδ T cells according to claim 5, wherein, The centrifugal force is 345~355g, and the time is 4~6min.

9. The method of isolating γδ T cells according to claim 5, wherein, Includes at least one of the following technical features: (1) The concentration of the first density gradient centrifugal medium is 35 vol%~45 vol% (2) The concentration of the second density gradient centrifugal medium is 70 vol%~75 vol%. (3) The volume ratio of the first density gradient centrifugation medium to the second density gradient centrifugation medium is 3~5:2; (4) The density gradient centrifugation speed is 2150~2250 r / min and the time is 18~22 min.

10. The use of the cell digestion solution according to any one of claims 1 to 4 in the preparation of a kit for isolating skin γδT cells.

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